Authors: Sophia Li & Payton Kujat
Research from the Gujral Lab
Introduction
Cancer is an extremely complex and a continuously evolving issue that researchers and scientists work on day and night. Metastasis is one of the many things in cancer
researchers continue to research. In this article we will explain metastasis and explore the differences and similarities between primary (original site) and metastasis tumors.
What is Metastasis?
Metastasis is the process of cancer cells leaving the primary tumor and traveling through the blood or lymphatic system to reach other organs and tissues in the body. Metastatic tumors originate from the primary tumor and form secondary tumors in other areas of the body. However, metastatic tumors contain different TME's (Tumor Microenvironment) than the primary tumor; thus, different tumors react differently to treatments. TME is the environment around a tumor, and the TME is heterogeneous, meaning various parts of the tumor have different cell types. Metastasis causes 90% of all cancer-related deaths due to the uniqueness of every metastatic tumor, showing the importance of further researching this topic.
Process of H & E Staining
To properly view our tumor samples, we performed a process known as H & E staining. This process stains the sample, allowing visualization of the structures, cells, and morphology.
We made a visual through the program BIORENDER of the steps we took to make this project possible (Fig. 1).
Figure 1
The first step is obtaining the tissue sample. Our samples consist of a primary colorectal tumor and a colorectal liver metastatic tumor, both taken from mice. The samples are fixed on a paraffin block, and a microtome (used to cut extremely thin slices of tissue) is used to slice the tissue samples. Next, we endured the process of H and E staining. The first step to H and E staining is to melt the sample in an oven at 60 Celsius for 30 minutes. Once melted, we placed it in xylene to dewax, then ethanol to rehydrate the tissue; we stained the nuclei with hematoxylin (stains the nuclei blue), rinsed, and used eosin to stain the cytoplasm. Afterward, we placed it in ethanol again to dehydrate and finished with xylene to re-wax. Finally, the sample was mounted and ready for viewing.
Using a microscope, we looked at the slides and identified the different structures of cells within the tumor.
Comparing and Defining the Structures Between Primary and Metastatic Tumors
The results of our H and E staining of the primary tumor are in Figure 2. The image contains red blood cells identified in the magenta-pink sections. In addition, it illustrates heterogeneity proven by the varying shades of purple of the various immune cells. The differing shades, textures, and fragmented nuclei are evidence of necrosis because the cancer cells are not getting enough blood to live.
As for the results of our H & E staining of liver metastasis (Figure 3), we observe an abundance of hepatocytes, cells residing in the liver. These cells contain large, round nuclei responsible for metabolism and detoxification. There is necrosis of cancer cells that have died due to injuries or lack of blood flow to the tissue. Sinusoids are small blood vessels that regulate blood flow within the liver between rows of hepatocytes. A system of macrophages called Kupffer cells resides within sinusoids. Kupffer are star-shaped and ingest bacterial pathogens that enter the liver.
Kupffer cells are native to the liver, and the early depletion of Kupffer cells can also facilitate tumor growth, defining them as tumor-associated macrophages (TAM), macrophages that support tumor growth instead of fighting against the tumor.
Figure 4
We compared the attributes of the primary and the secondary tumors (Fig. 4). Compared to the metastatic tumor, the primary tumor contains different immune cell functions and doesn't contain liver host cells. Whereas the liver metastatic contains liver host cells and has a heterogeneous TME. As for similarities, both tumors contain cancer cells and vasculature, signs of necrosis, and heterogeneity within the tumor.
Tumor-Associated-Macrophages
We will now specifically focus on the phenotype and morphology of the immune cell type called macrophages within primary and metastatic tumors. Research has shown that macrophages play a critical role in the tumor microenvironment. We want to understand the phenotype changes of macrophages in primary tumors and metastatic tumors. We performed a DAB staining to mark the macrophages in a brown color.
Aspect ratio is the measurement of macrophages stained brown (Figure 5 & 6). We use the aspect ratio to study the morphology and differences between macrophages within the primary and liver metastasis. We measured 50 macrophages from each tumor and discovered a significant difference in the average aspect ratio between the two tumors, showing that the macrophages in the primary are more elongated than in the liver metastasis. Although this was what our project concluded, further research has proven that most macrophages in metastatic tumors are more elongated than in primary tumors. This error in our research may have been due to involving multiple people in measuring the macrophages instead of having a singular person measure all the macrophages. We hypothesize this may have caused conflicting results.
We want to make sure we include correct information. From further research, we can confidently conclude that metastatic tumor macrophages are more elongated than primary tumor macrophages. Metastatic tumors contain elongated macrophages that actively promote cell proliferation and tissue repair. Our research project provides a notion that some samples of primary tumor macrophages are more elongated than liver metastasis macrophages. Further research must be done to properly prove this statement.
Overall, metastasis is a subject worth studying because there are still many mysteries and discoveries around the subject. Once a tumor begins metastasizing, it will continue to metastasize and grow tumors within the body. In most cases, treatment slows growth and reduces the symptoms of the metastasis. We hope that future research will bring forward a way not just to slow down metastasis, but to cure it completely.
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